Difference between revisions of "Team:Austin UTexas/Results"

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The do-it-yourself (DIY) movement is focused on making science more accessible to the public. Because many consumers brew their own kombucha, we have developed a set of DIY instructions that would allow an average person to analyze their home-brew and identify their kombucha’s species outside of a lab setting. This procedure is possible because of Gellan Gum, produced by the halobacterium Sphingomonas pauci-mobilis.
 
The do-it-yourself (DIY) movement is focused on making science more accessible to the public. Because many consumers brew their own kombucha, we have developed a set of DIY instructions that would allow an average person to analyze their home-brew and identify their kombucha’s species outside of a lab setting. This procedure is possible because of Gellan Gum, produced by the halobacterium Sphingomonas pauci-mobilis.
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Gellan Gum is produced through aerobic fermentation (Kang et al. 1982). This exopolysaccharide is a “high-molecular-mass, anionic polysaccharide which consists of a tetrasaccharide structure with 20% glucuronic acid, 60%glucose, and 20% rhamnose” (Wang et. al. 2006). The advantages of using Gellan in place of agar include: requiring half of the quantity, a consistent production, more clarity than agar, a reduced plate preparation time along with a faster setting time, stability at high temperatures, and lack of contamination factors found in agar that are toxic to some organisms (Ioannis et. al. 2007).
 
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Gellan Gum is produced through aerobic fermentation (Kang et al. 1982). This exopolysaccharide is a “high-molecular-mass, anionic polysaccharide which consists of a tetrasaccharide structure with 20% glucuronic acid, 60%glucose, and 20% rhamnose” (Wang et. al. 2006). The advantages of using Gellan in place of agar include: requiring half of the quantity, a consistent production, more clarity than agar, a reduced plate preparation time along with a faster setting time, stability at high temperatures, and lack of contamination factors found in agar that are toxic to some organisms (Ioannis et. al. 2007).
 
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This protocol involves creating media from every-day grocery store items, autoclaving or microwaving a culture of S. paucimobilis for sterilization, then pouring them into plates which can be streaked with microbes. <b>Figure 1</b> denotes each of the steps of the protocol. An S. paucimobilis colony is initially grown in conditions that focus the cell’s metabolism on multiplication. This maximizes the number of cells producing Gellan when inoculated into minimal media (Wu et. al. 2014).
 
This protocol involves creating media from every-day grocery store items, autoclaving or microwaving a culture of S. paucimobilis for sterilization, then pouring them into plates which can be streaked with microbes. <b>Figure 1</b> denotes each of the steps of the protocol. An S. paucimobilis colony is initially grown in conditions that focus the cell’s metabolism on multiplication. This maximizes the number of cells producing Gellan when inoculated into minimal media (Wu et. al. 2014).

Revision as of 02:22, 20 October 2016

Results


Click on one of the images below to learn more about our results!







Figure 4: amilCP expressed in E. coli and in liquid LB. Credit: Riya Sreenivasan

GOX Sequences as Putative Promoters

Three endogenous upstream regions of loci on the Gluconobacter oxydans chromosome were reported to show increased mRNA synthesis as pH decreased, were isolated and obtained, as seen in table 1 (Hanke, et al., 2012). Using Golden Gate assembly, these putative promoters have been placed on the Golden Gate entry vector pYTK001 for later use. By utilizing these pH-sensitive promoters with different reporters and transforming them into multiple organisms in kombucha, the visualization of the microbes and their location in kombucha would be possible (Lee, et al., 2015). This will serve as a stepping stone into further understanding how the microbiome of kombucha changes as it brews as well as determining organism concentration specific times during the brewing process.

Figure 5. Spun down P-atp2 constructs compared to controls in pH6-9. There is no clear gradient change in color expression. Credit: Ian Overman and Alex Alario

Table 1:The Three Endogenous GOX Sequences
Locus Tag Predicted Functions mRNA ratio pH4/pH6
GOX0647 Putative exporter protein, ArAE family 12.91
GOX0890 Hypothetical protein GOX0890 4.93
GOX1841 Hypothetical protein GOX1841 3.36

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References

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